Dual PDF Signaling Pathways Reset Clocks Via TIMELESS and Acutely Excite Target Neurons to Control Circadian Behavior Adam Seluzicki, Matthieu Flourakis, Elzbieta Kula-Eversole, Luoying Zhang ¤ , Valerie Kilman, Ravi Allada* Department of Neurobiology, Northwestern University, Evanston, Illinois, United States of America Abstract Molecular circadian clocks are interconnected via neural networks. In Drosophila, PIGMENT-DISPERSING FACTOR (PDF) acts as a master network regulator with dual functions in synchronizing molecular oscillations between disparate PDF(+) and PDF(2) circadian pacemaker neurons and controlling pacemaker neuron output. Yet the mechanisms by which PDF functions are not clear. We demonstrate that genetic inhibition of protein kinase A (PKA) in PDF(2) clock neurons can phenocopy PDF mutants while activated PKA can partially rescue PDF receptor mutants. PKA subunit transcripts are also under clock control in non-PDF DN1p neurons. To address the core clock target of PDF, we rescued per in PDF neurons of arrhythmic per 01 mutants. PDF neuron rescue induced high amplitude rhythms in the clock component TIMELESS (TIM) in per-less DN1p neurons. Complete loss of PDF or PKA inhibition also results in reduced TIM levels in non-PDF neurons of per 01 flies. To address how PDF impacts pacemaker neuron output, we focally applied PDF to DN1p neurons and found that it acutely depolarizes and increases firing rates of DN1p neurons. Surprisingly, these effects are reduced in the presence of an adenylate cyclase inhibitor, yet persist in the presence of PKA inhibition. We have provided evidence for a signaling mechanism (PKA) and a molecular target (TIM) by which PDF resets and synchronizes clocks and demonstrates an acute direct excitatory effect of PDF on target neurons to control neuronal output. The identification of TIM as a target of PDF signaling suggests it is a multimodal integrator of cell autonomous clock, environmental light, and neural network signaling. Moreover, these data reveal a bifurcation of PKA-dependent clock effects and PKA-independent output effects. Taken together, our results provide a molecular and cellular basis for the dual functions of PDF in clock resetting and pacemaker output. Citation: Seluzicki A, Flourakis M, Kula-Eversole E, Zhang L, Kilman V, et al. (2014) Dual PDF Signaling Pathways Reset Clocks Via TIMELESS and Acutely Excite Target Neurons to Control Circadian Behavior. PLoS Biol 12(3): e1001810. doi:10.1371/journal.pbio.1001810 Academic Editor: Justin Blau, New York University, United States of America Received August 1, 2013; Accepted February 5, 2014; Published March 18, 2014 Copyright: ß 2014 Seluzicki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The Ultima two-photon laser scanning microscope (Prairie Technologies) was supported by the National Institute of Neurological Disorders and Stroke (NINDS) NS054850. This work was funded by NINDS NRSA F31 NS065613-02 to A.S. and R01 NS059042 to R.A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: DD, constant darkness; FACS, fluorescence-activated cell sorting; GRASP, GFP reconstitution across synaptic partners; LD, light-dark; lLNv, large ventral-lateral neuron; PKA, protein kinase A; P-S, power-significance; sLNv, small ventral-lateral neuron. * E-mail: r-allada@northwestern.edu ¤ Current address: Department of Neurology, University of California San Francisco, San Francisco, California, United States of America Introduction Circadian clocks endow organisms with the ability to predict and respond adaptively to daily changes in the environment. In many taxa, these clocks consist of cell-autonomous molecular feedback loops, producing ,24-hour oscillations at the mRNA and protein levels. In insects and mammals these clocks are also connected in neural networks that stabilize and synchronize these molecular feedback loops and communicate timing information to regulate daily behavior. How network and cell-autonomous mechanisms collaborate to produce robust circadian rhythms remains a major question. In Drosophila, the molecular circadian clock consists of a set of interlocked transcriptional feedback loops in which the basic helix- loop-helix per-arnt-sim (bHLH-PAS) domain transcription factor CLOCK (CLK) forms a heterodimer with CYCLE (CYC) and binds E-boxes in the promoter regions of period (per), timeless (tim), vrille (vri), Par-domain protein 1e (Pdp1e) and clockwork orange (cwo), promoting their transcription (reviewed in [1]). PDP1e and VRI feed back to regulate the Clk and cryptochrome (cry) promoters [2,3], while CWO feeds back to regulate CLK/CYC activation at E- boxes [4–7]. PER and TIM proteins dimerize in the cytosol and are each required for their subsequent localization to the nucleus where PER inhibits CLK/CYC–mediated activation [8–14]. The CRY photoreceptor mediates light resetting via TIM degradation [15–18]. Clock function is evident as 24-h oscillations in the mRNA and protein levels of most of these clock components. The activity, stability, and subcellular localization of these proteins are largely controlled post-translationally by daily phosphorylation rhythms and subsequently by ubiquitin/proteasome dependent degradation [17,19–25]. In contrast to transcriptional regulators, significant oscillations have not been described for these post- translational regulators with the exception of the PP2A subunits tws and wdb [26]. In insects and mammals, intercellular signaling among pace- maker neurons in neural networks has been found to be critical for PLOS Biology | www.plosbiology.org 1 March 2014 | Volume 12 | Issue 3 | e1001810